www.aladdin-e.com Address:800 S Wineville Avenue, Ontario, CA 91761,USA Website:www.aladdin-e.com Email USA: [email protected] Email EU: [email protected] Email Asia Pacific: [email protected] Protocol of Protein Extraction Prepare ice! During the extraction procedure, the protein should always be kept on ice once it’s Each 6-well been put into the extraction buffer and later on in × 7 × 9 tissue plate order to avoid degradation and keep activity. Each M-PER Reagent 300 2100 2700 50 µl For fresh or frozen tissue µl µl µl 10% bleach, 100% ethanol, dH2O Protease and Homogenizer Phosphatase 3 µl 21 µl 27 µl 0.5 µl Inhibitor 1. Prepare 10% bleach, 100% ethanol, dH2O for cleaning the Homogenizer. No more than 3 Prepare dry ice. Take out the eppendorf 5ml/tube. containing the tissues from -80°C, put a pieces of tissue with a pipette tip into the plastic tube For 6 samples, prepare 2+6+2=10 tubes of each containing the extraction reagent. Put the cleaning solution. 10× 5ml=30ml. eppendorf on the dry ice after use. For 8 samples, prepare 2+8+2=12 tubes of each cleaning solution. 12× 5ml=60ml. 4 Homogenize tissues Set the homogenizer to 30. First wash, each Twice (i.e. 10% bleach→10% Wash as step1 mentioned. → → → → bleach 100% ethanol 100% ethanol H2O Homogenize each sample for about 20-30 H2O). seconds. (Remember to wash the homogenizer after each sample homogenizing.) Between each sample, wash once (i.e. 10% bleach→100% ethanol→ H2O). 5 Transfer to eppendorf tubes and incubate on Last wash, each Twice (i.e. 10% bleach→10% ice for 10 minutes. bleach→100% ethanol→100% ethanol→ H2O→ (Meanwhile, put the eppendorf containing the H2O). tissues back to -80°C.) For cell line (Step1, 2 then go to step 6) 6 Spin at 4°C, 13000rpm for 10 min. 1 Carefully removes culture medium. Wash the 7 Prepare the sample buffer in the hood. cell with PBS twice gently. Usually 250 ul buffer / tissue sample. Add 1 ml PBS into the well, scrap the cell from Usually 50 ul buffer / tissue sample. the plate. (For 100 μl: 95ul of Laemmli Sample buffer (Bio- Spin, RT or 4°C, 3000rpm, 1-3 min. Rad, blue, store at RT) + 5 ul of β-ME). 2. Prepare extraction reagent. (The same reagent for cell line protein extaction) 1 www.aladdin-e.com For tissue Each l Aliquot into labeled eppendorf. 100 250 ul × 6 × 8 × 10 l Laemmli 9. Add same volume of supernatant into the Sample 95 237.5 1425 1900 2375 eppendorf with the sample buffer. buffer β-ME 5 12.5 75 100 125 10. Incubate at >70°C (70-100°C) at heat block for 5 min. (Remember to cover the tube cap) Each For cell 11. Store the samples at -80°C. line sample 100 50 ul × 6 × 8 × 10 l Laemmli For More information on related products, Sample 95 47.5 285 380 475 please click: Aladdin buffer β-ME 5 2.5 15 20 25 2 .